1. Field of the Invention
The present invention relates to a modified TXNIP (thioredoxin-interacting protein) protein, a method for preparing the modified TXNIP protein, a polynucleotide encoding the modified protein, an expression vector comprising the polynucleotide, a transformant introduced with the expression vector, a method for crystallizing a modified TRX(thioredoxin)-TXNIP complex using the modified TXNIP protein, and a method for screening a substance regulating interaction between TRX and TXNIP, an inhibitor of TRX activity, and a substance regulating TXNIP function, using the crystal structure of the TRX-TXNIP complex mutein.
2. Description of the Related Art
TRX, which is up-regulated in many cancers, is involved in a wide range of cell signaling processes comprising cellular immune response, in response to a variety of oxidative stresses, and thus TRX is an important protein that has received much attention from the world's major pharmaceutical companies as an attractive target for anticancer drugs (Powis, G. et al., 2007 Current Opinion in Pharmacology 7:392, Mukherjee, A. et al., 2008 The British Journal of Radiology 81:S57). TXNIP, the only currently known endogenous inhibitor of TRX, inhibits interaction between TRX and a range of proteins involved in cell signaling as well as redox-potential activity of TRX. Therefore, studies have been actively conducted on TRX-TXNIP roles in cancers and metabolic diseases (Schulze, P. C. et al., 2004 J Biol Chem 279:30369, Dunn, L. L. et al., 2010 Arteriosclerosis, Thrombosis, and Vascular Biology 30:2089, Spindel, O. N. et al., 2012 Antioxidants & Redox Signaling 16:587), which is supported by the experimental results that TXNIP is strongly down-regulated in a variety of tumor cells and tissues (Shin, K. H. et al., 2008 Biochem Biophys Res Commun 372:880), and TXNIP knockout mice have an increased incidence of hepatocellular carcinoma (Sheth, S. S. et al., 2006 Oncogene 25:3528). The function of TXNIP as the tumor suppressor inhibiting TRX activity is well-known in the research related to inhibition of TRX-ASK1 (apoptosis signal-regulating kinase 1) interaction by TXNIP. That is, TXNIP-TRX interaction inhibits TRX-ASK1 interaction, leading to increased levels of reactive oxygen species (ROS) and promotion of cell apoptosis by ASK1 (Matsuoka, S. et al., 2008 Cancer Science 99:2485).
In addition to its function as an endogenous inhibitor of TRX, TXNIP is correlated with glucose levels (Muoio, D. M. 2007 Cell Metabolism 5:412, Parikh, H. et al., 2007 PLoS Medicine 4:2158). Glucose stimulates TXNIP transcription through a carbohydrate-response element present in the TXNIP promoter (Minn, A. H. et al., 2005 Endocrinology 146:2397) and its association with transcription factors MLX (max-like protein X) and MondoA (Stoltzman, C. A. et al., 2008 Proc Natl Acad Sci USA 105:6912). An elevated level of TXNIP has led to a reduction in the number of pancreatic beta-cells, insulin secretion, and peripheral glucose uptake (Parikh, H. et al., 2007 PLoS Medicine 4:2158, 23). By contrast, TXNIP deficiency protected against beta-cell apoptosis, and enhanced insulin sensitivity (Yoshihara, E. et al., 2010 Nature Communications 1:127, Chen, J. et al., 2008 FASEB Journal 22:3581). Despite considerable efforts to identify TXNIP characteristics and molecular mechanism of TRX regulation by TXNIP which are involved in metabolic diseases as well as cancers, understanding of TXNIP and regulation mechanism of TXNIP-TRX has reached a limit, because of a lack of their structural information.
Recent studies change their approach from a random approach to exploration of a number of anticancer drug candidates and therapeutic candidates to a new approach to development of effective therapeutic agents by exploration of target proteins, investigation of structure and reaction mechanism of the target proteins, and design and development of drug candidates through more efficient and scientific protein engineering. To design and develop drug candidates, investigation of their three-dimensional structures is essential. To investigate the three-dimensional structures, the priority is to acquire the protein in a stable form. However, the three-dimensional structure of the TXNIP protein, for all its importance, has not been revealed yet, because the TXNIP protein is a redox protein containing 11 cysteines, making it difficult to handle during production.
Accordingly, the present inventors have made many efforts to investigate the three-dimensional structure of the TXNIP protein. As a result, the present inventors have established a method for preparing a stable TRX-TXNIP complex having a purity of 90% or more, through various types of engineering, and they prepared a complex crystal of a TRX protein and a modified TXNIP protein, activity of which has no significant difference from that of the wild-type and demonstrated interaction between TRX and TXNIP at the protein level. Consequently, they have found that a substance capable of regulating the activities of TRX and TXNIP or interaction there between can be developed, based on the protein structure, thereby completing the present invention.